WO2019117648A1

WO2019117648A1 - Biosensor

Info

Publication number: WO2019117648A1
Application number: PCT/KR2018/015852
Authority: WO
Inventors: 전진우; 황혜진; 박연수; 김기범
Original assignee: (주)플렉센스
Priority date: 2017-12-13
Filing date: 2018-12-13
Publication date: 2019-06-20

Abstract

The present invention relates to a biosensor. The biosensor according to the present invention comprises: a detection structure which is formed in the shape of a plate and has an immobilized material disposed on either side or both sides thereof and binding specifically to a target material.

Description

Biosensor

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosensor technique for immunoassay, and more particularly, to a biosensor structured to improve immunoassay speed and sensitivity.

An enzyme-linked immunosorbent assay (ELISA) is an analytical technique that detects and quantifies target substances such as peptides, proteins, antibodies and hormones. In ELISA, a target material, such as an antigen, etc., is immobilized on a solid surface and then complexed with a reagent, e.g., an antibody polymerized with an enzyme. When the substrate is added, the substrate reacts with the enzyme to generate a measurable reaction product, so that the target substance can be detected and analyzed by evaluating the enzyme activity. The types of ELISAs include direct ELISA, indirect ELISA, sandwich ELISA, and competitive ELISA.

Direct ELISA is detected by immobilizing the antigen on the surface of a multi-well plate and reacting specifically with antibodies conjugated with HRP or other markers.

In the indirect ELISA, similarly to the direct ELISA, the antigen is immobilized on the surface of the multi-well plate. Here, after the primary antibody specifically reacts with the antigen, the labeled secondary antibody reacts with the primary antibody and is detected . This method can be used to detect a specific antibody in a serum sample by replacing the primary antigen with a serum.

In a sandwich ELISA (or sandwich immunoassay), two antibodies specific for an antigen are used. One of the antibodies acts as a capture antibody coated on the surface of the multi-well plate to immobilize the antigen, and the other antibody is polymerized with the antigen to facilitate the detection of the antigen.

In the case of competitive ELISA, also referred to as inhibition ELISA or competitive immunoassay, the concentration of the antigen is measured by signal interference. The antigen in the sample competes with the reference antigen for binding to the labeled antibody, where the reference antigen is preferentially coated on the multiple well plate. Since the sample is pre-reacted with the labeled antibody and then added to the well, depending on the amount of the antigen in the sample, the antibody capable of binding to the reference antigen may be more or less. According to this, as the number of antigens in the sample increases, the signal is weakened as the reference antigen is detected less and the signal becomes stronger as the amount of the antigen in the sample becomes smaller as more antigens are labeled in the well.

As described above, the present invention can improve the concentration of the reactant or antibody by improving the hook effect, which is known as a disadvantage of the conventional ELISA, and it is possible to improve the convenience of the immunoassay technique remarkably, And to provide a biosensor that is remarkably shortened.

A biosensor according to the present invention includes a detection structure formed in a plate shape and having a fixation substance that is specifically bound to a target material disposed on at least one of a surface and a surface of the biosensor.

In addition, in the biosensor according to the present invention, at least one of the one surface and the other surface of the detection structure is formed in a protrusion shape, and a nanostructure is formed on the outer surface to which the fixing substance is bonded.

Also, in the biosensor according to the present invention, the target substance may be an amino acid, a peptide, a polypeptide, a protein, a glycoprotein, a lipoprotein, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a sugar, a carbohydrate, an oligosaccharide, , Hormones, metabolites, cytokines, chemokines, receptors, neurotransmitters, antigens, allergens, antibodies, substrates, metabolites, cofactors, inhibitors, drugs, drugs, nutrients, plion, toxins, poisons, At least one selected from the group consisting of chemical agents, biological agents, bacteria, viruses, radioactive isotopes, vitamins, heterocyclic aromatic compounds, carcinogens, mutagen, drugs, amphetamines, barbiturates, hallucinogens, It is more than one.

Further, in the biosensor according to the present invention, the immobilizing material and the target substance react with each other when the detecting structure is immersed in the cuvette containing the sample containing the target substance.

Further, in the biosensor according to the present invention, the biosensor further includes a grip portion connected to one end of the detection structure and gripped by a user.

Further, in the biosensor according to the present invention, the cap further includes a detachable cap inserted into the inlet of the cuvette, connecting the detection structure and the grip portion to each other.

The biosensor according to the present invention further includes a fixing part which is disposed on an outer surface of the cap and which is brought into close contact with the inner circumferential surface of the cuvette by a restoring force generated when the cap is deformed when the cuvette is inserted into the cuvette do.

Further, in the biosensor according to the present invention, the detecting structure is divided into a dipping part immersed in the sample and a non-dipping part, and the non-dipping part is provided with a narrow width part whose width is smaller than the width of the dipping part do.

In the biosensor according to the present invention, at least one or more of the narrow-pore portions are formed concavely on at least one of both side surfaces of the detection structure, and are formed along the length direction of the detection structure.

Further, in the biosensor according to the present invention, the detection structures are arranged in a plurality of spaced apart from each other.

Further, in the biosensor according to the present invention, the biosensor further includes a pair of guards disposed opposite to each other with the detection structure therebetween to protect the detection structure.

Further, in the biosensor according to the present invention, a body having a predetermined length and a plurality of recesses are formed from one surface of the body so as to accommodate a sample containing the target substance, and the detection structure And at least one sensor strip including a reaction chamber in which the sensor strip is disposed.

Further, in the biosensor according to the present invention, the sensor strip may be detachably attached to one surface of the biosensor.

Further, in the biosensor according to the present invention, the detection structures are arranged vertically in multiple stages in a plurality of spaced apart from each other.

Further, the biosensor according to the present invention may further include a sample injection port formed to be recessed from one surface of the body so as to communicate with the reaction chamber.

According to another aspect of the present invention, there is provided a biosensor, further comprising: an insertion protrusion protruding from one surface of the fixing plate, wherein an insertion hole is formed in the main body such that the insertion protrusion is inserted or penetrated, And is attached to the fixing plate.

Further, in the biosensor according to the present invention, a corner of one end of the main body is recessed toward the inside, and when the insertion projection is inserted into the insertion hole, And a fixing jaw which is spaced apart from the fixing plate and protrudes from one surface of the fixing plate.

The features and advantages of the present invention will become more apparent from the following detailed description based on the accompanying drawings.

Prior to that, terms and words used in the present specification and claims should not be construed in a conventional and dictionary sense, and the inventor may properly define the concept of the term in order to best explain its invention It should be construed as meaning and concept consistent with the technical idea of the present invention.

The biosensor according to the present invention can relatively increase the concentration of the receptor or antibody that reacts per unit volume, thereby enhancing the convenience of immunoassay, shortening the analysis time, and further improving the sensitivity of the reaction.

FIG. 1 (A) is a conceptual diagram showing a conventional ELISA analysis method using sequential reaction, and FIG. 1 (B) is a conceptual diagram showing a one-step ELISA analysis method using simultaneous reaction.

2A to 2C are side views schematically showing a detection structure of a biosensor according to an embodiment of the present invention.

3A to 3E are perspective views schematically illustrating a nanostructure of a biosensor according to an embodiment of the present invention.

FIGS. 4A and 4B are cross-sectional views of a cuvette-type biosensor according to an embodiment of the present invention, and FIG. 4C is a perspective view.

5 is a front view showing a process of inserting the biosensor shown in FIG. 4C into the cuvette.

FIG. 6 is a side view showing a state where the biosensor shown in FIG. 4C is inserted into the cuvette.

7 is a perspective view of a cuvette type biosensor according to another embodiment of the present invention.

8 is a perspective view of a strip-type biosensor according to an embodiment of the present invention.

9 is a perspective view showing a detection structure of a strip biosensor according to an embodiment of the present invention.

10 is a perspective view illustrating a sensor strip of a strip-type biosensor according to an embodiment of the present invention.

11 is a perspective view of a strip-type biosensor according to another embodiment of the present invention.

12 to 14B are an exploded perspective view and an assembled perspective view of a strip-type biosensor according to another embodiment of the present invention.

15 is a flowchart of a sample analysis method using a cuvette type biosensor according to the present invention.

16 is a flowchart of a sample analysis method using a strip biosensor according to the present invention.

FIGS. 17A to 17F are graphs showing experimental results of absorbance measured according to the concentration of various detection target antibodies using the biosensor according to the present invention. FIG.

FIGS. 18A and 18B are graphs showing experimental results obtained by measuring the absorbance of a reaction product of an antibody to be detected having a different concentration using a biosensor having different numbers of detection structures according to the present invention. FIG.

BRIEF DESCRIPTION OF THE DRAWINGS The objectives, specific advantages and novel features of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: FIG. It should be noted that, in the present specification, the reference numerals are added to the constituent elements of the drawings, and the same constituent elements are assigned the same number as much as possible even if they are displayed on different drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the following description of the present invention, detailed description of related arts which may unnecessarily obscure the gist of the present invention will be omitted.

Regardless of the type of ELISA, a common challenge for faster and more sensitive ELISAs is to enhance the sensing signal of the target material at a faster rate of reaction. Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The factors that determine the reactivity in immunoassays vary, but the concentration of the protein in the sample and the concentration of the receptor or antibody that reacts with the protein are the most important factors.

In the case of a general ELISA, since the reaction area of the microtiter plate for immunoassay is limited to the area for accommodating the sample, the concentration of the receptor or antibody involved in the reaction per unit volume of the same sample is limited. In addition, in the case of the conventional ELISA technique for increasing the surface-to-volume ratio for the purpose of increasing the sensitivity, diffusion of various reagents and / or analytes is reduced, resulting in a problem that the reaction time is excessively increased. A biosensor according to the invention has been developed.

1 (A), in a general sandwich ELISA, a reaction complex is produced by reacting a sample containing a target substance such as protein on a substrate on which a receptor or an antibody is immobilized, and after washing the reaction complex The marker is reacted with the polymerized detection antibody. This sequential reaction process is very complex. Therefore, there is a problem that an experienced user must perform in the laboratory and takes more than 4 hours to analyze.

1 (B), in order to overcome the excessive analysis time and complexity of the reaction process of the sandwich ELISA shown in FIG. 1 (A), in some immunoassay techniques, a sample containing a target substance such as protein A method in which the marker-conjugated detection antibody is firstly mixed and reacted, and the mixture is reacted with the receptor or the substrate on which the antibody is immobilized is used. This method is very convenient because it can perform immunoassay with one sample injection, and the analysis time is shortened about 8 times compared with the conventional method. However, the effectiveness of such an immunoassay technique may not be realized due to various reasons. For example, when a large amount of target protein is present, the unreacted target protein that has not reacted with the marker-conjugated detection antibody reacts with the antibody immobilized on the substrate. This phenomenon is referred to as a "hook effect" I call it. In addition, the concentration can not be accurately identified due to the unreacted target protein without the marker. In order to solve this problem, the concentration of the receptor or antibody which is fundamentally involved in the reaction should be higher than the target protein.

감도 증강 나노구조 활성 표면을 구비한 바이오센서Biosensor with Sensitivity Enhanced Nanostructured Active Surface

In the biosensor, if the active surface area of the detection structure for fixing biomolecules and / or analytes (organic or inorganic substances) is increased, the density of immobilized biomolecules and / or analytes can be increased, Can be expected. In this case, it is desirable to increase the active surface area while reducing the negative influence of the diffusion transport of various biomolecules, reagents and / or analytes.

Accordingly, the biosensor according to the present invention has an active surface area including a textured or modified polymer surface. Here, a submicron or a nanostructure may be formed on the surface of the polymer. Hereinafter, the submicron structure may be a structure in which at least one of physical dimensions such as length, width, height, diameter, and the like is about 1 탆 or less, and a nanostructure is a structure having at least one physical dimension of about 100 nm or less Respectively.

FIGS. 2A to 2C are side views schematically showing a detection structure of a biosensor according to an embodiment of the present invention. FIGS. 3A to 3E are perspective views schematically showing a nanostructure of a biosensor according to an embodiment of the present invention. to be.

As shown in FIGS. 2A to 2C, the biosensor according to the present invention includes a plate-shaped detection structure 10 having one surface and a surface opposite to the other surface. Here, an active surface may be included on each of the one side and the other side, and the active surface may be embodied as a textured polymer surface having a submicron or nanostructure 11. The submicron or nanostructure 11 may be formed of a polymeric material, for example, the same material as that of the detection structure 10. Here, the active surface means a surface on which one or more biomolecules and / or analytes are immobilized for immunoassay. The active surface may be implemented by chemical treatment to specifically bind biomolecules and / or analytes, such as antibodies or antigens.

The detection structure 10 may have a planar structure. A biomolecule or a reagent, such as a fixing material C, for example, an antibody, may be disposed on at least one surface of a flat plate surface, that is, one surface or another surface. Here, the fixing substance (C) is a substance that specifically binds to the target substance. On the other hand, the target substance may be provided as a stand-alone substance or may be contained in a sample, and the detection substance (for example, biomolecule or reagent) in which the marker is polymerized may be further included in the sample. The target material provided may be an amino acid, peptide, polypeptide, protein, glycoprotein, lipoprotein, nucleoside, nucleotide, oligonucleotide, nucleic acid, sugar, carbohydrate, oligosaccharide, polysaccharide, fatty acid, lipid, hormone, metabolite, cytokine, chemokine , A receptor, a neurotransmitter, an antigen, an allergen, an antibody, a substrate, a metabolite, a cofactor, an inhibitor, a drug, a drug, a nutrient, a plion, a toxin, a poison, an explosive, an insecticide, At least one selected from viruses, radioactive isotopes, vitamins, heterocyclic aromatic compounds, carcinogens, mutagen, drugs, amphetamines, barbiturates, hallucinogens, wastes, and contaminants.

Antibodies, proteins, peptides, Deoxyribo Nucleic Acid (DNA), and the like are examples of the immobilizing substance (C), which is specifically determined according to the target substance. ), A synthetic reagent comprising a ribonucleic acid (RNA), a peptide nucleic acid (PNA), an enzyme, an enzyme substrate, a hormone, a hormone receptor and a functional group, Combinations thereof.

The marker may be any one of HRP (horseradish peroxidase), basic alkaline phosphatase, and fluorescein.

The sample is a reagent which reacts with the above-mentioned markers, such as ABTS (2,2'-azinobis [3-ethylbenzothiazoline-6-sulfonic acid] -diammonium salt) or TMB (3,3 ', 5,5'- Tetramethylbenzidine). &Lt; / RTI >

However, the target substance, the fixing substance (C), the marker, and the reagent are not necessarily limited to the above-mentioned kinds.

3A to 3E, the submicron or nanostructure 11 may be formed on at least one of the one surface and the other surface of the detection structure 10. [ Here, the submicron or nanostructure 11 may be formed on one or both surfaces of the detection structure 10 and / or on the entire surface of the other surface, and on the outer surface of the submicron or nanostructure 11, (C) may be disposed. The detection structure 10 having the submicron or nanostructure 11 has a larger surface area than the surface of the flat sensing structure 10 without the submicron or nanostructure 11, So that the substance C reacts with the target substance disposed in the detection structure 10.

On the other hand, the submicron or nanostructure 11 may be formed in at least one protrusion shape. The submicron or nanostructure 11 may include protrusions whose cross-sectional area gradually decreases as the distance from the base increases. These projections improve the surface area at which the immobilizing material C is fixed, and at the same time reduce the negative influence of the diffusion transport of the various reagents and / or analytes. In addition, the protrusions may be in the form of spheres, pyramids, triangles, rectangles, cubes, plates, discs, cylinders, wires, rods, sheets, And may be formed in the form of a fractal or the like, and the predetermined region may be cut or twisted in the above form.

For example, the protrusions may be formed with a cut spherical shape, such as a hemisphere shape, wherein the hemispherical protrusions are regularly arranged to form a submicron or nanostructure array. At this time, the submicron or nanostructure arrays may be arranged so that the projections are arranged in a zigzag shape, and the adjacent projections are in contact with each other (see FIG. 3A).

3B) or a semicircular shape (see FIG. 3C), and a pyramid having a smaller cross-sectional area in a direction perpendicular to the surface of the detection structure 10 A pyramidal frustrum (see FIG. 3D), or a conical frustrum (see FIG. 3E).

Since the submicron or nanostructure 11 improves the surface area, the concentration of the receptor or antibody involved in the reaction can be relatively increased. Thus, the sensitivity of the submicron or nanostructure 11 is improved compared to the conventional ELISA assay, and the concentration of the receptor or antibody It is possible to effectively control the hook effect that occurs when a higher concentration of protein is present.

On the other hand, the submicron or nanostructure arrays can be arranged regularly, for example, periodically, on the submicron or nanostructure 11. However, the present invention is not limited thereto, and the submicron or nanostructure 11 may be irregularly arranged, or irregularly arranged in one direction and irregularly arranged in the other direction. On the other hand, the submicron or nanostructure 11 may include nanowires, nanopillars, or nanofibers.

감도 및 시약 확산 증강 Sensitivity and reagent diffusion enhancement 큐벳형Cuvet type 바이오센서 Biosensor

The biosensor according to the present invention can be provided in a cuvette type and a strip type. The structure will be described below in detail. As described above, it is preferable that the biosensor has a structure for detecting a structure that reduces the negative influence on the diffusion transport of various biomolecules, reagents, and / or analytes while simultaneously increasing the active surface. In order to meet such a demand, the biosensor according to the present invention may include a transparent container having at least one cavity and a plurality of active surfaces inserted into the cavity to immobilize biomolecules or reagents. Here, the biosensor includes a transparent detection structure inserted into the cavity, and the transparent detection structure may include at least one main surface providing at least one active surface. Many active surfaces increase the active surface area that fixes biomolecules and / or analytes. In addition, each of the active surfaces is spaced apart from one another at predetermined intervals to facilitate diffusion transport of various biomolecules, reagents, and / or analytes in the ELISA. In the present invention, the distance between the adjacent active surfaces may be approximately 300 to 9000 탆 so as not to interfere with the diffusion transport. In addition, the total active area provided for immobilizing the various biomolecules and / or analytes used in the ELISA may be from 1.0 to 6.0 mm2 / l per unit volume. In this range, the detectable concentration of the analyte is increased by at least 2 times, more than 20 times, compared with the sensor not having the transparent detection structure, at which time the reaction rate does not decrease. However, the space and the area of the active region are determined depending on the shape of the biosensor, the target material, and the like, and thus the present invention is not limited thereto.

FIGS. 4A and 4B are cross-sectional views of a cuvette type biosensor according to an embodiment of the present invention, FIG. 4C is a perspective view, FIG. 5 is a front view showing a process of inserting the biosensor shown in FIG. 6 is a side view showing a state in which the biosensor shown in FIG. 4C is inserted into the cuvette.

4A, the cuvette-type biosensor 1000A according to the first embodiment of the present invention includes a cuvette 1, and all or a part of the inner surface of the cuvette 1 is detected by a detection structure And a fixing substance C, which reacts with the target substance in the sample 3, may be disposed thereon. The submicron or nanostructure 11 may be formed on the inner surface area of the cuvette 1 provided as a detection structure and the immobilizing material C may be disposed on the submicron or nanostructure 11.

Referring to FIG. 4B, the cuvette-type biosensor 1000B according to the second embodiment of the present invention is implemented in such a manner that the detection structure 10 is inserted into a cuvette. That is, the cuvette-type biosensor 1000C is configured such that the detecting structure 10 is inserted into the cuvette 1 containing the target material and the fixing material C disposed on the detecting structure 10 reacts with the target material. Here, the target material may be included in the sample 3 and be accommodated in the cuvette 1. At this time, when the detection structure 10 is immersed in the sample 3, the immobilizing material C reacts with the target material . On the other hand, the fixing structure C is disposed on the detection structure 10, and the nanostructure 11 is formed on the entire surface of the detection structure 10 and / And the fixing material C may be disposed on the outer surface. In one embodiment, a submicron or nanostructure 11 may be formed on the detection structure 10. At this time, the submicron or nanostructure 11 may be formed on at least a part of the active region of each main surface of the detection structure 10. [ In another embodiment, the sub-micron or nanostructure 11 may be formed on one surface and / or the entire surface of the other surface of the detection structure 10 so that the entire surface thereof serves as an active region. Alternatively, the submicron or nanostructure 11 may be formed on only one side and / or a part of the other side of the detection structure 10, and the active area may be provided only for a part thereof.

Similar to the cuvette type biosensor 1000A (see Fig. 4A) described above, the cuvette 1 may be provided with a fixation material C in all or a part of the inner surface thereof, The submicron or nanostructure 11 may be formed in the region and the immobilizing material C may be disposed on the submicron or nanostructure 11.

The absorbance of the detection structure 10 and the cuvette 1 itself can be measured using the absorbance of the detection structure 10 as a reference absorbance.

The detection structure 10 and the cuvette 1 may be formed of a material such as polycarbonate, polyethylene terephthalate, polymethylmethacrylate, triacetylcellulose, cyclic olefin, polyarylate, polyacrylate, Polyethylene naphthalate, polybutylene terephthalate, polyimide, or the like. However, this is only an example, and there is no particular limitation in the case of a light-transmitting material. The material of the detecting structure 10 and / or the cuvette 1 is suitably selected in consideration of whether the material is effective for forming an active surface through chemical modification or functionalization of the surface, or is capable of direct modification to the surface. For example, the submicron or nanostructure 11 can be formed integrally with a solid substrate such as a polymer substrate, or can be formed separately on the substrate or can be formed by a suitable method such as etching or plasma treatment . Thus, in one embodiment, the active surface may comprise a biomolecule and / or a polymeric surface that is provided directly as an active surface to which the analyte is immobilized. In this embodiment, the active surface does not include any additional material, such as metal, but in other embodiments may further comprise additional material to form an active surface.

Meanwhile, the cuvette-type biosensor 1000B according to the present invention may further include a grip portion 20 connected to one end of the detection structure 10. The grip portion 20 is a member formed to be gripped by a user and is connected to one end of the detection structure 10. The user can hold the grip section 20 and insert the cuvette 1 from the free end of the detection structure 10 so that the detection structure 10 can be immersed in the sample 3. The free end of the detecting structure 10 means the opposite end of the one end of the detecting structure 10 connected to the holding portion 20. [

Here, one or more detection structures 10 may be provided. When a plurality of detecting structures 10 are provided, each of the detecting structures 10 is connected to and fixed to one holding portion 20. At this time, one of the detecting structures 10 and one or the other of the detecting structures 10 are opposed to each other. And can be arranged in parallel and spaced apart from each other by a predetermined distance. Here, the interval between adjacent detection structures 10 may be any of the above-described separation distances, which is an important factor for achieving such effects as an increase in optical density and / or a reduction in reaction time. When the plurality of detecting structures 10 are provided, the fixing material is disposed at a high density per unit area, so that the sensitivity of the sensor can be improved and the hook effect can be controlled.

In addition, the cuvette-type biosensor 1000B according to the present invention may further include a cap 30. Here, the cap 30 is removably inserted into the inlet of the cuvette 1 and is formed to close the inlet of the opened cuvette 1. At this time, the entire area can be closed by the cap 30 at the inlet of the cuvette 1, but only a part of the opening may be closed. The cap 30 is disposed below the grip portion 20 and connects the grip portion 20 and the detection structure 10 to each other and is fixed in contact with the inner surface of the cuvette 1, Do not move in the cuvette (1). In FIG. 4B, the grip portion 20 and the cap 30 are shown as one body, but they may be formed to have a separate structure as shown in FIG. 4C.

Referring to FIGS. 4C to 6, the cuvette-type biosensor 1000C according to the third embodiment of the present invention includes all the technical features of the cuvette-type biosensor 1000B described above, As described above in the biosensor 1000B, duplicate matters are omitted or simply described. In the following, a cuvette type biosensor 1000C will be described focusing on the differences.

A clearance may be formed between the outer surface of the cap 30 and the inner surface of the inlet of the cuvette 1 depending on the size of the inlet of the cuvette 1 in the cuvette type biosensor 1000B according to the present embodiment, Is not fixed to the cuvette 1, so that it is difficult to analyze the sample 3 accurately. The biosensor 1000C according to the present embodiment may further include the fixing portion 40 so that the detecting structure 10 is fixed regardless of the relative size between the cuvette 1 and the cap 30. [

The fixing portion 40 is disposed on the outer surface of the cap 30 so that the original position or shape of the cap 30 is changed when the cap 30 is inserted into the cuvette 1, And is formed to be in close contact with the inner peripheral surface. When the fixing portion 40 disposed on the cap 30 is thus brought into close contact with the cuvette 1, the detecting structure 10 connected to the cap 30 is fixed in the cuvette 1.

Specifically, when the cap 30 is inserted into the cuvette 1, the fixing portion 40 is deformed while being pressed by the inner surface of the cuvette 1, and is pressed against the inner surface of the inlet of the cuvette 1 by the elastic force As shown in Fig. The fixing portion 40 may use the elasticity of the material itself such as rubber or the like, or may use the properties of parts such as a spring. However, the fixing portion 40 does not necessarily have to use the elastic force of the material or the component, but can be implemented through a predetermined structure, which will be described in detail below.

The fixing portion 40 may extend from the outer surface of the cap 30 and may be bent in a predetermined direction. For example, the cap 30 may extend outward from the outer surface of the cap 30 and may be bent in parallel with the outer surface of the cap 30 to form an "a" 1 can be formed in a structure in which the protrusions can be closely contacted by the tension while being pressed. At this time, since the fixing portion 40 is pressed and moved in the direction of the cap 30, the facing portion of the outer surface of the cap 30 facing the fixing portion 40 can be recessed.

In addition to the above-described structure, the fixing portion 40 may extend on the inner surface of the recessed portion and may have a protruding portion protruding outside the outer surface of the cap 30 so as to have a "C" shape.

As a result, the fixing portion 40 can be deformed into various structures other than the above-described structure as long as the cap 30 is brought into close contact with the inner surface of the cuvette 1 by tension when the cap 30 is inserted into the cuvette 1. [

Meanwhile, the detection structure 10 of the biosensor 1000C according to the present embodiment may include the narrow portion 12. When the detection structure 10 is immersed in the sample 3, it is divided into a dipping portion immersed in the sample 3 and a non-dipping portion not immersed. In the non-dipping portion, (12) is formed.

In order to analyze the sample 3, the detection structure 10 is inserted into the cuvette 1. At this time, the gap between the detection structure 10 and the inner surface of the cuvette 1 or the gap between the detection structures 10 A capillary force is generated and the sample 3 rises. Due to the rise of the sample (3), the amount of the sample (3) necessary for the analysis is increased, and the analytical reliability is seriously degraded. The narrow part (12) is a solution to this problem. The sample 3 rises along the detection structure 10 by the attractive force acting between the detection structure 10 and the sample 3. When the width of the sample 3 is narrowed at the narrow portion 12, Becomes smaller, and the sample 3 no longer rises.

Specifically, the narrow portion 12 may be formed in the shape of an up-and-down preventing groove 17 recessed in the side surface of the detecting structure 10. Since the rising preventing groove 17 is recessed by a predetermined depth from one side of the detecting structure 10 to the other side of the detecting structure 10, The width, that is, the distance between the both side surfaces is short. These rising preventing grooves 17 may be formed on only one side of the detecting structure 10, but may be formed on both sides of the detecting structure 10. In the case where the lift preventing grooves 17 are formed on the both side surfaces, they may be formed so as to face each other, but the present invention is not limited thereto and may be staggered in a zigzag fashion. Also, the rising preventing grooves 17 may be formed along the side surface of the detecting structure 10, spaced apart from each other by a predetermined distance in the longitudinal direction, and a plurality of the rising preventing grooves 17 may be formed.

The rising preventing groove 17 may be curved to be rounded, but it is not necessarily formed in such a shape, but it may be recessed in any form as long as the width of the detecting structure 10 is narrowed .

7 is a perspective view of a cuvette type biosensor according to another embodiment of the present invention. As shown in FIG. 7, the biosensor 1000D according to the fourth embodiment of the present invention includes the above- In addition to the biosensor 1000C according to the example, the guard structure 50 may further include a pair of guards 50 to protect the detection structure 10. Here, the pair of guards 50 is a member that is disposed opposite to the detection structure 10, with the detection structure 10 therebetween. The guards 50 are arranged such that a plurality of detecting structures 10 are arranged between the pair of guards 50 even when a plurality of detecting structures 10 are arranged. Since the guards 50 are disposed on both outermost sides of the detection structure 10, the detection structure 10 is prevented from contacting the inner surface of the cuvette 1, and the detection structure 10 is protected from external impact . The guard 50 may be formed in a plate shape, but the shape of the guard 50 is not limited thereto. However, when the guard 50 is formed in the shape of a plate, the sample 3 may rise in the gap between the detection structure 10 arranged side by side or the inner surface of the cuvette 1, The narrow portion 12a may be formed to have a relatively narrow width at the height. At this time, the narrow portion 12a may also be formed by recessing the lift preventing groove 17a on the side surface of the guard 50. The narrow portion 12a may be formed in the guard 50, It is not necessary that the narrow portion 12a be formed.

Further, the fixing material may be bonded to at least one of the one surface and the other surface of the guard 50, thereby improving the density of the fixing material per unit volume.

2A to 7, the thickness of the detection structure 10 may be selected in the range of about 100 to 5,000 mu m, and at least one main surface of the detection structure 10, that is, For example, one side and / or the other side may have an area of 10 to 100 mm < 2 >. Also, the active surface may account for 30-100% of the main surface. The size of the cuvette 1 and the detection structure 10 may be such that the active surfaces are spaced apart from each other by a suitable distance, The size of the cuvette 1 can be determined so that about 1 to about 20 detection structures 10 are inserted.

감도 및 시약 확산 증강 Sensitivity and reagent diffusion enhancement 스트립형Strip type 바이오센서 Biosensor

Hereinafter, a strip biosensor will be described. In this embodiment, the optical transparent container accommodating the at least one detection structure includes a strip container having a plurality of cavities. In the above-described cuvette type biosensor, the main surface of at least one plate-like detecting structure is arranged so as to be opposed to each other in a direction parallel to the depth direction in the cavity, while the present strip type biosensor has at least one plate- , And the main surfaces are arranged so as to face each other perpendicularly to the depth direction of the cavity. A strip biosensor according to an embodiment of the present invention includes a plate structure having a main surface facing each other in parallel to each other. Here, the main surface faces the bottom surface of the cavity, is arranged substantially parallel to the bottom surface, and can be spaced apart from each other by an interval of about 500 mu m.

On the other hand, the transparent detection structure may include a plate structure in which the center portion is connected to the inside of the cavity at the central portion (see Figs. 8 to 11). Here, the main surfaces of at least one or more of the transparent detecting structures may overlap each other in a direction perpendicular to the depth direction of the cavity. On the other hand, when two or more transparent detection structures are arranged, the mutually facing main surfaces of two adjacent transparent detection structures may be spaced apart by an interval of about 500 탆 or more.

FIG. 8 is a perspective view of a strip biosensor according to an embodiment of the present invention, FIG. 9 is a perspective view schematically showing a detection structure of a strip biosensor according to the present invention, Type biosensor according to the present invention.

8 to 10, the strip-type biosensor includes a main body 150 having a predetermined length, and a plurality of main bodies 150, which are recessed from one surface of the main body 150 to accommodate a sample containing the target material, And at least one sensor strip (100) including a reaction chamber (130) in which a dog is formed and in which a detection structure (10) is disposed.

The strip type biosensor according to the present invention includes a sensor strip 100. The sensor strip 100 includes a reaction chamber 130 in a plate-shaped body 150 having a predetermined length and width, And the detection structure 10 is disposed in the reaction chamber 130.

The reaction chamber 130 is recessed from one of the outer surfaces of the main body 150 so that the sample is filled therein, and a plurality of the reaction chamber 130 are arranged along the longitudinal direction of the main body 150. On the other hand, a fixing material may be disposed on the bottom surface of the reaction chamber 130, and a protruding nano structure (not shown) similar to that described above may be formed on the bottom surface of the reaction chamber 130. By arranging the fixing material, the density of the fixing material per unit volume can be increased.

The detection structure 10 is disposed in each of the plurality of reaction chambers 130 so as to be immersed in the sample contained in the reaction chamber 130. Here, the surface of the detection structure 10 may be formed in a planar shape, or a submicron or nanostructure 11 may be additionally formed on at least one or at least one surface of at least one of the one surface and the other surface, The material can be placed (see FIG. 9).

At least one sensor strip 100 may be provided and a plurality of reaction chambers 130 and detection structures 10 are provided in each of the sensor strips 100 so that a plurality of samples can be simultaneously analyzed. That is, analysis of different contents is performed for the same sample, or different capture bodies are fixed for each of the reaction chambers 130 or for each detection structure 10 in the same reaction chamber 130, The sample can be analyzed. On the other hand, the other surface opposite to one surface of the sensor strip 100 having the opening of the reaction chamber 130 may be disposed on the fixing plate 200.

The fixing plate 200 is formed in a plate shape having a predetermined width and thickness, and at least one sensor strip 100 is detachably attached to one surface of the plate. At this time, the fixing plate 200 and the sensor strip 100 can be detached and attached by the insertion protrusion 400 and the insertion hole 120. The insertion hole 120 may be formed in a shape corresponding to the external shape of the insertion protrusion 400 so as to be inserted or fixed such that the insertion protrusion 400 is inserted and released. The inserting protrusion 400 protrudes from one side of the fixing plate 200 and the inserting hole 120 is formed on the other side of the main body 150 of the sensor strip 100 so that the sensor strip 100 and the fixing plate 200 can be detached and attached. At this time, the insertion protrusion 400 may be formed on the sensor strip 100, and the insertion hole 120 may be formed on the fixing plate 200.

Since the biosensor according to the present invention analyzes the target material through the measurement of the absorbance, external light must be irradiated to the detection structure 10, and the fixation plate 200 may have the puncture 210. The perforations 210 are holes penetrating through the thickness direction of the fixing plate 200 and the reaction chamber 130 is disposed on a region of the fixing plate 200 where the perforations 21 are formed. Therefore, the reaction chamber 130 and the perforations 21 may be provided in the same number in positions corresponding to each other. The bottom surface of the reaction chamber 130 and the detection structure 10 are also made of a material capable of transmitting light, for example, a bottom surface of the reaction chamber 130 and a detection structure 10, And may be made of a polymeric material such as polycarbonate, polyethylene terephthalate, polymethylmethacrylate, triacetylcellulose, cyclic olefin, polyarylate, polyacrylate, polyethylene naphthalate, polybutylene terephthalate or polyimide . However, this is an embodiment, and the material is not necessarily limited thereto.

In addition, the sensor strip 100 according to the present embodiment may include a plurality of

detection structures

10, 10a, 10b, and 10c. Here, the plurality of

detection structures

10, 10a, 10b, and 10c may be vertically arranged in a multi-stage spaced apart from each other at a predetermined interval along the depth direction of the reaction chamber 130. [ At this time, any one detection structure 10a and the

other detection structure

10b or 10c may be arranged to face each other. By providing a plurality of the detecting structures 10 in this way, the density (concentration) of the fixed substance per unit volume is increased. At this time, a submicron or nanostructure 11 may be formed on the surface of the detection structure 10, and a fixing material may be disposed on the outer surface of the submicron or nanostructure 11.

In addition, the strip biosensor according to the present embodiment may further include a sample injection port 300. Since the sample injection port 300 is recessed from one surface of the main body 150 so as to communicate with the reaction chamber 130, the sample injected through the sample injection port 300 flows into the reaction chamber 130, The structure 10 is immersed in the sample.

11 is a perspective view of another embodiment of the strip-type biosensor according to the present invention.

11, the strip-type biosensor according to the present embodiment may further include a fixing jaw 500 for more firmly fixing the sensor strip 100 to the fixing plate 200. As shown in FIG. The fixing jaws 500 are formed to protrude from one surface of the fixing plate 200 and are spaced apart from the insertion protrusions 400 on the fixing plate 200 at a predetermined interval. The spacing distance between the fixing protrusions 400 and the fixing protrusions 500 is set such that when the inserting protrusions 400 are inserted into the insertion holes 120, It is determined to contact the outer surface. The sensor protrusion 400 may be inserted into the insertion hole 120 and may be fixed to the recessed corner of the sensor strip 100. In this case, The sensor strip 100 can be firmly fixed to the fixing plate 200 while the jaws 500 are in close contact with each other.

12 to 14B are an exploded perspective view and a combined perspective view of a strip biosensor according to another embodiment of the present invention.

As shown in FIGS. 12 to 14B, one or more detection structures according to an embodiment of the present invention may be formed in a protruding shape extending from the inner surface of the reaction chamber.

FIG. 12 shows an example for implementing the shape of the detection structure. Referring to FIG. 12, the sensor strip 100 in which the detection structure 10 is disposed is formed by stacking a plurality of plates. The plate includes a bottom plate 110a in the form of a flat plate, a spacer plate 110b having a perforation in the center thereof, and a sensor plate 110c having a perforation in the central portion thereof and a protrusion extending in the central direction from the inner surface of the perforation And the bottom plate 110a, the spacer plate 110b, and the sensor plate 110c are sequentially stacked and assembled to form the strip type biosensor according to the present invention. At this time, the reaction chamber 130 is formed in a state where the spacer plates 110b and the sensor plate 110c are formed at positions corresponding to each other, and the protrusion of the sensor plate 110c is connected to the

detection structure

10 , 10a, and 10b.

On the other hand, two or more sensor plates 110c may be stacked. At this time, the protrusions of one of the first sensor plates 111c and the protrusions of the other of the second sensor plates 113c may be formed so as not to overlap each other when they are stacked. In order to realize this, when the depth direction of the reaction chamber 130 is the Z axis, the protrusion of the first sensor plate 111c protrudes in the X axis direction perpendicular to the Z axis, and the protrusion of the second sensor plate 113c The protruding portion can be deflected at a predetermined angle in the Y-axis direction with respect to the X-axis direction. In this case, the protruding portions of the plurality of

sensor plates

111c and 113c to be stacked are staggered from each other along the depth direction of the reaction chamber, and the

detection structures

10a and 10b are arranged in a pattern corresponding thereto. However, the arrangement pattern of the protrusions with respect to the depth direction of the reaction chamber 130 is not necessarily arranged so as to be staggered as described above, but the protrusions of the plurality of

sensor plates

111c and 113c are formed at the same position, And may be formed to overlap with each other along the depth direction of the chamber 130.

Here, the projections of the sensor plate 110c may be spaced apart from each other by two or more along the inner circumferential surface of the perforation. In one example, the spacing between the protrusions of the sensor plate 110c may be spaced 30 to 180 degrees about the perforation. However, the spacing can be changed according to the number of protrusions, the circumference of the inner circumference of the perforations, and the like. In this case, the projections of one sensor plate 110c and the other sensor plate 110c may be disposed at intervals between the protrusions of the

sensor plates

110c and 113c so that they do not overlap with each other.

On the other hand, the two or more sensor plates 110c can be continuously stacked, and furthermore, at least one spacer plate 110b can be stacked therebetween. Also, two or more spacer plates 110b may be stacked between the bottom plate 110a and the sensor plate 110c, and one or more spacer plates 110b may be continuously stacked on the sensor plate 110c. In this case, the capacity of the reaction chamber 130 can be increased, and the inner peripheral surface of the perforation of each of the spacer plates 110b can be used as the active surface, so that the biomolecule and / or analyte, reagent and / The diffusion of the substance can be improved.

On the other hand, any one of the distal ends of the protrusions extends from the inner circumferential surface of the perforation, but the opposite free end is spaced apart from the inner circumferential surface of the perforation, and is also spaced from the free end of the other protrusions. Accordingly, the central portion of the reaction chamber 130 surrounded by the protrusions preferably arranges the protrusions so that the pipette can be easily accommodated.

In the strip biosensor according to the present invention, two or more reaction chambers 130 may be formed by providing two or more perforations at positions corresponding to each other with respect to the spacer plate 110b and the sensor plate 110c, Protrusions may be formed for each perforation of the plate 110c so that the detection structure 10 may be disposed in each of the reaction chambers 130. [ In this case, with reference to Figs. 13A to 13B, a strip biosensor is formed in each of a plurality of reaction chambers, in which a plurality of detection structures are arranged so as to overlap each other along the depth direction of the reaction chamber.

On the other hand, as shown in Figs. 14a to 14b, by stacking sensor plates having different arrangements of projections, a strip-type biosensor is arranged in each of a plurality of reaction chambers so that a plurality of detection structures are staggered along the depth direction of the reaction chamber .

감도 및 확산 증강 센서 조립체를 이용한 면역분석 방법Immunoassay method using sensitivity and diffusion enhancement sensor assembly

The biosensor according to the present invention can be effectively used for various immunoassays such as ELISA. An ELISA assay method according to one embodiment prepares an ELISA kit according to any of the embodiments of the present invention. Here, the ELISA kit comprises one or more ELISA reagents, analytes and / or biomolecules, and a sensor assembly suitable for ELISA.

In addition, the ELISA assay may further comprise performing an ELISA reaction in a transparent container such as a cuvette or a sensor strip with one or more reaction chambers.

In addition, the ELISA assay method comprises the steps of: preparing a solution containing a target substance and a marker polymerization detection reagent specifically binding to the target substance; Immobilizing a capture reagent that is specifically bound to the target material on the active surface of the sensor assembly; Immersing at least a portion of the active surface in the solution so that the target material is specifically bound to the capture reagent and the marker polymerization detection reagent; And detecting the target substance specifically bound to the capture reagent and the marker polymerization detection reagent.

On the other hand, an ELISA assay method according to another embodiment comprises preparing an ELISA well, wherein the ELISA well comprises a transparent container and at least one enhancement layer in the transparent container, And the bonding surface area ratio of the enhancement layer per liquid volume may be 1.0 to 5.0 mm 2 / ㎕. In addition, the method may further comprise performing an ELISA reaction with the transparent container, wherein one washing step may be performed.

The ELISA protocol and / or components according to the present invention are intended for use in indirect ELISA, streptavidin detection direct ELISA, sandwich ELISA, competition ELISA and / or strep-biotin detection sandwich ELISA. An ELISA kit according to the present invention comprises at least one of a coating buffer, a blocking buffer (e.g. PBS with 1% BSA), and a wash buffer (e.g. PBS with 0.05% v / v Tween-20) . The ELISA kit may further comprise a substrate solution (e.g., TMB Core + (BHU062) or pNPP (BUF044)) and a stop solution (e.g., 0.2 MH ₂ SO ₄ or 1 M NaOH).

Meanwhile, the ELISA assay method according to the present invention comprises the steps of coating a well with an antigen solution, optionally washing the plate with distilled water, adding a blocking buffer and washing the plate, adding the secondary antibody polymerized with the enzyme Washing the plate, adding the substrate solution and generating the reaction, and measuring the absorbance in the cuvette or well. This method can be applied to indirect ELISA.

On the other hand, an ELISA assay method according to the present invention comprises the steps of coating a well, optionally washing the plate with distilled water, adding a blocking buffer and washing the plate, adding the sample to the well, Adding the antibody to each well (optionally including a washing step), adding the enzyme-conjugated streptavidin to the well (which may optionally include a washing step), adding the substrate solution to the well Or adding it to a cuvette, and measuring the absorbance. This method can be applied to direct ELISA.

Meanwhile, a direct ELISA according to the present invention comprises (i) coating a solid support with an antigen dissolved in a coating buffer; (ii) reacting the solid support with the blocking reagent for 1 hour to block non-specific binding sites of the solid support; (iii) washing the solid support with PBS or PBST three times for 1 minute; (iv) reacting a solid support with a first detection agent that binds to the antigen; (v) washing the solid support 5 times with PBS or PBST for 1 minute to remove the nonspecifically bound first detection reagent; And (vi) detecting a combined first detection reagent using a detection system such as UV, fluorescence, chemiluminescence, or other detection method. The first detection reagent may be a detection reagent connected to a fluorescent dye or a reporter enzyme such as basic alkaline phosphatase (AP) or HRP (horseradish peroxidase), though it is not particularly limited. This detection reagent converts a colorless substrate to a colored product and the optical density of the colored product can be measured with an ELISA plate reader at the target wavelength.

An indirect ELISA according to the present invention comprises (i) coating a solid support with an antigen dissolved in a coating buffer; (ii) reacting the solid support with the blocking reagent for 1 hour to block non-specific binding sites of the solid support; (iii) washing the solid support with PBS or PBST three times for 1 minute; (iv) reacting the solid support with a first detection agent in solution for 1 hour; (v) washing the solid support with PBS or PBST three times for 1 minute to remove the nonspecifically bound first detection reagent; (vi) reacting the second detection reagent in solution with the solid support for 1 hour; (vii) washing the solid support 5 times with PBS or PBST for 1 minute to remove the nonspecifically bound second detection reagent; And (viii) detecting the combined second detection means using a detection system such as UV, fluorescence, chemiluminescence, or other detection methods. Here, the second detection time is associated with the first detection time. The second detection reagent is not particularly limited, but may be a detection reagent linked to a reporter enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP). This detection reagent converts a colorless substrate to a colored product and the optical density of the colored product can be measured with an ELISA plate reader at the target wavelength.

A direct ELISA according to the present invention comprises a first reaction step between a solid support and an antigen; A second reaction step between the solid support and the blocking reagent; And a third reaction step between the solid support and the first detection reagent. The reaction step may be a two-phase reaction and may involve a coupling reaction between the antigen on the solid support and the detection reagent.

An indirect ELISA according to the present invention comprises a first reaction step between a solid support and an antigen; A second reaction step between the solid support and the blocking reagent; A third reaction step between the solid support and the first detection reagent, and a fourth reaction step with the solid support and the second detection reagent. The reaction step may be a two-phase reaction and may involve a coupling reaction between the antigen on the solid support and the detection reagent.

In a direct ELISA according to the present invention, the first reaction step (antigenic coating) may be allowed to react for at least 2 hours and the other reaction step may be allowed to react for about 1 hour.

In the indirect ELISA according to the present invention, the first reaction step (antigenic coating) can be allowed to react for at least 2 hours and the other reaction step for about 1 hour.

The cell-based ELISA (C-ELISA) according to the present invention is a method for the detection and quantification of cell proteins, in which the cell proteins are subjected to post-translational modifications related to cellular activities (for example, phosphorylation and degradation) -translational modification. Cells are plated, treated according to the experimental requirements, fixed directly to the wells, and then permeabilized. After permeabilization, the immobilized cells can be subjected to similar procedures as conventional immunoblots, i.e., blocking, reaction with the first antibody, washing, reaction with the second antibody, addition of a chemilumescent substrate, have.

The ELISA according to the present invention was prepared by coating active wells and antigens for 4 days at 4 DEG C for 1 day, blocking the wells at 37 DEG C for 2 hours, then performing antibody and polymer binding at 37 DEG C for 2 hours each, After performing the enzyme substrate reaction at room temperature, the absorbance can be measured.

The ELISA kit according to the present invention can be used as an ELISA kit such as Acetylcholine ELISA Kit, AGE ELISA Kit, CXCL13 ELISA Kit, FGF23 ELISA Kit, HMGB1 ELISA Kit, iNOS ELISA Kit, LPS ELISA Kit, Malondialdehyde ELISA Kit, Melatonin ELISA Kit, An OVA ELISA Kit, an Oxytocin ELISA Kit, a PGE2 ELISA Kit, a PTHrP ELISA Kit, an S100b ELISA Kit, a Tenascin C ELISA Kit, a VEGF-B ELISA Kit, and a Versican ELISA Kit.

Hereinafter, a one-step antigen detection immunoassay using the biosensor according to the present invention will be described.

FIG. 15 is a flowchart of a sample analysis method using a cuvette type biosensor according to the present invention, and FIG. 16 is a flowchart of a sample analysis method using a strip type biosensor according to the present invention.

15, a one-step antigen detection immunoassay using a cuvette type biosensor according to the present invention comprises: (a) a sample containing a target substance (for example, antigen); and Preparing a detection material polymerization solution (for example, a detection antibody complex solution and the like) each containing a detection substance (for example, detection antibody, etc.) ) Immersing the detection structure in which the immobilization material capable of binding to the target substance (for example, a capture antibody, etc.) is bonded to the surface in one of the sample and the detection substance polymerization solution, Or immersing the sample in a mixed solution of the sample and the detection material polymerization solution, and (c) measuring the absorbance.

In the step (b), the fixed substance, the target substance, and the detection substance polymerized with the marker react with each other. After about 15 to 30 minutes have elapsed, the detection structure is immersed in a cuvette containing an enzyme substrate, and absorbance is measured in that state. At this time, after completion of the step (b), the detection structure may be further washed to remove the detection target material on which the unreacted target substance and / or the marker is polymerized, and the detection structure may be immersed in the cuvette containing the enzyme substrate.

On the other hand, in the case of the strip-type biosensor, in step (b), either one of the sample and the detection material polymerization solution is first injected through the sample injection port and then another one is injected, or the sample and the detection substance The polymer solution is mixed and injected, and the absorbance can be measured by injecting the enzyme substrate in step (c).

According to the above-mentioned method, aflatoxin B1, streptomycin, human epididymis protein 4 (HE4), carcinoembryonic antigen (CEA), mouse IgG and Cortisol were selected as target substances, , And the results are shown in Figs. 17A to 17F.

When analyzing grain mycotoxins (see FIG. 17A), detection was possible in the concentration range of 19.53-312.5 pg / mL and the detection time was 45 minutes. In the case of pesticide residues of fruit trees (see FIG. 17b), the detection was carried out in a concentration range of 0.39 to 50 ng / mL and the detection time was 30 minutes.

In order to confirm that the sensitivity of the biosensor according to the present invention is improved, analysis was also performed using a competitor's biosensor for HE4, CEA, Mouse IgG, and Cortisol. The ELISA kit, which is a biosensor of a competitor, was used. The absorbance measured by the biosensor according to the present invention is indicated by a solid line, and the absorbance measured by a competitor's biosensor is indicated by a dotted line.

In the HE4 assay (see FIG. 17C), the biosensor of the present invention was detected within a concentration range of 6.1 to 390 pg / mL within 30 minutes, while a competitor's biosensor was in a concentration range of 78 to 5,000 pg / mL Detection time was 4 hours.

In the case of CEA analysis (see FIG. 17D), the biosensor according to the present invention was detectable in a concentration range of 0.2 to 390 ng / mL, and the detection time was about 15 to 30 minutes. On the other hand, competitive biosensors were detected in the concentration range of 1-65 ng / mL for 90 minutes.

17A), the detection range of the biosensor according to the present invention is 0.05 to 25 ng / mL and the detection time is 30 minutes, while the detection range of the competitor's biosensor is 7.8 to 500 ng / mL, and the time required was 120 minutes.

When using a biosensor according to the present invention, it was possible to detect within a concentration range of 0.05 to 25 ng / mL within 30 minutes. However, when a competitive biosensor is used, 7.8 to 500 ng / mL for 120 minutes.

Comprehensively analyzing the above results, it can be seen that when the biosensor according to the present invention is used, the sensitivity of the sensor is greatly improved and the time required for detecting the target substance is shortened.

As described above, the active or reactive region of the biosensor according to the present invention is relatively much wider compared to conventional ELISA plates. Compared to the conventional ELISA plate, the biosensor according to the present invention can react with a larger number of immobilizing substances (capture molecules to receptors) with the target substance. As a result, the biosensor according to the present invention can reduce the hook effect, thereby improving the assay sensitivity, and at the same time, increasing the reaction rate and reducing the reaction time.

Referring to FIG. 18A, when the biosensor according to the present invention is used (marked), the sensitivity of the assay is increased as compared with the case of using a competitor's microtiter plate (96 well) .

In addition, by stacking a plurality of detection structures according to the present invention, the concentration of the receptor and the antibody reacting with the protein such as antigen and the like is substantially increased. For example, referring to FIG. 18B, a biosensor having six detection structures according to the present invention detects IgG with high sensitivity as compared with the case where two detection structures are provided. In general, the sensitivity of the biosensor having a plurality of detection structures is a result of a fixed substance (for example, capture molecules to receptors) being arranged at a high density and increasing their diffusion.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the present invention. It is obvious that the modification or improvement is possible.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

The biosensor according to the present invention is a plate-shaped detection structure in which an active surface is formed on one surface and / or the other surface, respectively, thereby increasing the convenience of immunoassay, shortening the analysis time and further enhancing the sensitivity of the reaction The possibility of industrial use is recognized.

Claims

A detecting structure formed in a plate shape and having a fixing material that is specifically bonded to a target material on at least one of a surface and a surface of the substrate;

.
The method according to claim 1,

Wherein at least one of the one surface and the other surface of the detection structure is formed in a protrusion shape, and a nanostructure to which the fixation substance is bonded is formed on the outer surface.
The method according to claim 1,

Wherein the target material is selected from the group consisting of amino acids, peptides, polypeptides, proteins, glycoproteins, lipoproteins, nucleosides, nucleotides, oligonucleotides, nucleic acids, sugars, carbohydrates, oligosaccharides, polysaccharides, fatty acids, lipids, hormones, metabolites, cytokines, Receptor, neurotransmitter, antigen, allergen, antibody, substrate, metabolite, cofactor, inhibitor, drug, drug, nutrient, plion, toxin, poison, explosive, insecticide, chemical agonist, biohazard agent, bacteria, virus Wherein the biosensor is at least one selected from the group consisting of radioactive isotopes, vitamins, heterocyclic aromatic compounds, carcinogens, mutagen, drugs, amphetamines, barbiturates, hallucinogens, wastes, and contaminants.
The method according to claim 1,

Wherein the fixation substance and the target substance react with each other in a state that the detection structure is inserted and immersed in a cuvette containing a sample containing the target substance.
The method of claim 4,

A gripper connected to one end of the detecting structure and gripped by a user;

Further comprising a biosensor.
The method of claim 5,

A cap connected to the detection structure and the grip portion so as to be removably inserted into an inlet of the cuvette;

Further comprising a biosensor.
The method of claim 6,

A fixing part which is disposed on an outer surface of the cap and which is brought into close contact with an inner circumferential surface of the cuvette by a restoring force generated when the cap is deformed when the cuvette is inserted into the cuvette;

Further comprising a biosensor.
The method of claim 4,

The detection structure

An immersion part immersed in the sample and a non-immersion part,

And the narrowed portion in the non-stuck portion is narrower in width than the width of the immersed portion.
The method of claim 8,

The narrow-

And at least one of both side surfaces of the detection structure is recessed and formed,

Wherein at least one biosensor is formed along the longitudinal direction of the detection structure.
The method of claim 4,

The detection structure

A plurality of biosensors spaced apart from one another and arranged in parallel.
The method according to claim 1,

At least one sensor including at least one sensor including a reaction chamber in which a plurality of the reaction chamber are disposed inside the reaction chamber, the reaction chamber being recessed from one surface of the body so as to receive a sample containing the target substance, strip;

Further comprising a biosensor.
The method of claim 11,

A fixing plate on which the sensor strip is detachably attached;

Further comprising a biosensor.
The method of claim 11,

The detection structure

A plurality of biosensors spaced apart from one another and arranged vertically in multiple stages.
The method of claim 11,

A sample injection port formed to be recessed from one surface of the main body so as to communicate with the reaction chamber;

Further comprising a biosensor.
The method of claim 12,

An insertion protrusion protruding from one surface of the fixing plate;

Further comprising:

Wherein the inserting hole is formed in the body so that the insertion protrusion is inserted or penetrated, and the sensor strip is attached to the fixing plate.
16. The method of claim 15,

A corner of one end of the main body is depressed toward the inside,

A fixing jaw that is spaced apart from the insertion protrusion and protrudes from one surface of the fixing plate so as to come into contact with a corner of one end of the main body when the insertion protrusion is inserted into the insertion hole;

Further comprising a biosensor.